RESUMO
Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.
Assuntos
Acetilglucosaminidase , Mucopolissacaridose III , Humanos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Acetilglucosaminidase/metabolismo , Acetilglucosaminidase/antagonistas & inibidores , Acetilglucosaminidase/química , Acetilglucosaminidase/genética , Sítio Alostérico/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacosRESUMO
Highly penetrant autosomal dominant Alzheimer's disease (ADAD) comprises a distinct disease entity as compared to the far more prevalent form of AD in which common variants collectively contribute to risk. The downstream pathways that distinguish these AD forms in specific cell types have not been deeply explored. We compared single-nucleus transcriptomes among a set of 27 cases divided among PSEN1-E280A ADAD carriers, sporadic AD, and controls. Autophagy genes and chaperones clearly defined the PSEN1-E280A cases compared to sporadic AD. Spatial transcriptomics validated the activation of chaperone-mediated autophagy genes in PSEN1-E280A. The PSEN1-E280A case in which much of the brain was spared neurofibrillary pathology and harbored a homozygous APOE3-Christchurch variant revealed possible explanations for protection from AD pathology including overexpression of LRP1 in astrocytes, increased expression of FKBP1B, and decreased PSEN1 expression in neurons. The unique cellular responses in ADAD and sporadic AD require consideration when designing clinical trials.
Assuntos
Doença de Alzheimer , Presenilina-1 , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Humanos , Presenilina-1/genética , Masculino , Feminino , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Análise de Sequência de RNA/métodos , Autofagia/genética , Transcriptoma , Idoso , Neurônios/metabolismo , Neurônios/patologia , Pessoa de Meia-Idade , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação a Tacrolimo/genética , Idoso de 80 Anos ou mais , Análise de Célula ÚnicaRESUMO
J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.
Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico , Humanos , Adenosina Trifosfatases/metabolismo , Ácido Edético , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares/químicaRESUMO
The male gamete is a highly differentiated cell that aims to fuse with the oocyte in fertilization. Sperm have silenced the transcription and translational processes, maintaining proteostasis to guarantee male reproductive health. Despite the information about the implication of molecular chaperones as orchestrators of protein folding or aggregation, and the handling of body homeostasis by the endocannabinoid system, there is still a lack of basic investigation and random controlled clinical trials that deliver more evidence on the involvement of cannabinoids in reproductive function. Besides, we noticed that the information regarding whether recreational marijuana affects male fertility is controversial and requires further investigation. In other cell models, it has recently been evidenced that chaperones and cannabinoids are intimately intertwined. Through a literature review, we aim to explore the interaction between chaperones and cannabinoid signaling in sperm development and function. To untangle how or whether this dialogue happens within the sperm proteostasis. We discuss the action of chaperones, the endocannabinoid system and phytocannabinoids in sperm proteostasis. Reports of some heat shock and lipid proteins interacting with cannabinoid receptors prove that chaperones and the endocannabinoid system are in an intimate dialogue. Meanwhile, advancing the evidence to decipher these mechanisms for introducing innovative interventions into routine clinical settings becomes crucial. We highlight the potential interaction between chaperones and cannabinoid signaling in regulating proteostasis in male reproductive health.
Assuntos
Canabinoides , Proteostase , Endocanabinoides/metabolismo , Sementes , Chaperonas Moleculares/metabolismo , Espermatozoides/metabolismo , Canabinoides/metabolismoRESUMO
Plants trigger endoplasmic reticulum (ER) pathways to survive stresses, but the assistance of ER in plant tolerance still needs to be explored. Thus, we selected sensitive and tolerant contrasting abiotic stress sorghum varieties to test if they present a degree of tolerance to ER stress. Accordingly, this work evaluated crescent concentrations of tunicamycin (TM µg mL-1): control (0), lower (0.5), mild (1.5), and higher (2.5) on the initial establishment of sorghum seedlings CSF18 and CSF20. ER stress promoted growth and metabolism reductions, mainly in CSF18, from mild to higher TM. The lowest TM increased SbBiP and SbPDI chaperones, as well as SbbZIP60, and SbbIRE1 gene expressions, but mild and higher TM decreased it. However, CSF20 exhibited higher levels of SbBiP and SbbIRE1 transcripts. It corroborated different metabolic profiles among all TM treatments in CSF18 shoots and similarities between profiles of mild and higher TM in CSF18 roots. Conversely, TM profiles of both shoots and roots of CSF20 overlapped, although it was not complete under low TM treatment. Furthermore, ER stress induced an increase of carbohydrates (dihydroxyacetone in shoots, and cellobiose, maltose, ribose, and sucrose in roots), and organic acids (pyruvic acid in shoots, and butyric and succinic acids in roots) in CSF20, which exhibited a higher degree of ER stress tolerance compared to CSF18 with the root being the most affected plant tissue. Thus, our study provides new insights that may help to understand sorghum tolerance and the ER disturbance as significant contributor for stress adaptation and tolerance engineering.
Assuntos
Sorghum , Tunicamicina/farmacologia , Sorghum/genética , Chaperonas Moleculares , Retículo Endoplasmático , Estresse do Retículo EndoplasmáticoRESUMO
The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.
RESUMO
Mychael Lourenco is an Assistant Professor of Neuroscience at the Institute of Medical Biochemistry Leopoldo de Meis, Federal University of Rio de Janeiro. Research in his lab focusses on understanding the molecular mechanisms underlying cognitive impairment in neurodegeneration and his research on Alzheimer's disease has been recognized by many awards both in Brazil and internationally. He serves as a Reviews Editor for the Journal of Neurochemistry and led this special issue on Brain Proteostasis as a Guest Editor. Here we interviewed him to hear his thoughts on the future of neuroscience and on career development and training.
Assuntos
Neuroquímica , Proteostase , Encéfalo , BrasilRESUMO
Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins.
Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Bacillus thuringiensis/genética , Insetos , Larva/genética , Chaperonas Moleculares , Proteínas de Choque Térmico HSP90/genética , Peptídeo Hidrolases , Proteínas de Choque Térmico HSP70/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidadeRESUMO
The Unfolded protein response (UPR), triggered by stress in the endoplasmic reticulum (ER), is a key driver of neurodegenerative diseases. GM2 gangliosidosis, which includes Tay-Sachs and Sandhoff disease, is caused by an accumulation of GM2, mainly in the brain, that leads to progressive neurodegeneration. Previously, we demonstrated in a cellular model of GM2 gangliosidosis that PERK, a UPR sensor, contributes to neuronal death. There is currently no approved treatment for these disorders. Chemical chaperones, such as ursodeoxycholic acid (UDCA), have been found to alleviate ER stress in cell and animal models. UDCA's ability to move across the blood-brain barrier makes it interesting as a therapeutic tool. Here, we found that UDCA significantly diminished the neurite atrophy induced by GM2 accumulation in primary neuron cultures. It also decreased the up-regulation of pro-apoptotic CHOP, a downstream PERK-signaling component. To explore its potential mechanisms of action, in vitro kinase assays and crosslinking experiments were performed with different variants of recombinant protein PERK, either in solution or in reconstituted liposomes. The results suggest a direct interaction between UDCA and the cytosolic domain of PERK, which promotes kinase phosphorylation and dimerization.
Assuntos
Gangliosidoses GM2 , Doença de Sandhoff , Animais , Atrofia , Gangliosidoses GM2/metabolismo , Neuritos/metabolismo , Doença de Sandhoff/terapia , Ácido Ursodesoxicólico/farmacologia , eIF-2 Quinase/metabolismoRESUMO
Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.
Assuntos
Proteínas Ferro-Enxofre , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ferro/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
The function of chaperones is to correct or degrade misfolded proteins inside the cell. Classic molecular chaperones such as GroEL and DnaK have not been found in the periplasm of Yersinia pseudotuberculosis. Some periplasmic substrate-binding proteins could be bifunctional, such as OppA. Using bioinformatic tools, we try to elucidate the nature of the interactions between OppA and ligands from four proteins with different oligomeric states. Using the crystal structure of the proteins Mal12 alpha-glucosidase from Saccharomyces cerevisiae S288C, LDH rabbit muscle lactate dehydrogenase, EcoRI endonuclease from Escherichia coli and THG Geotrichum candidum lipase, a hundred models were obtained in total, including five different ligands from each enzyme with five conformations of each ligand. The best values for Mal12 stem from ligands 4 and 5, with conformation 5 for both; for LDH, ligands 1 and 4, with conformations 2 and 4, respectively; for EcoRI, ligands 3 and 5, with conformation 1 for both; and for THG, ligands 2 and 3, with conformation 1 for both. The interactions were analyzed with LigProt, and the length of the hydrogen bridges has an average of 2.8 to 3.0 Å. The interaction within the OppA pocket is energetically favored due to the formation of hydrogen bonds both of OppA and of the selected enzymes. The Asp 419 residue is important in these junctions.
Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Proteínas Periplásmicas de Ligação , Yersinia pseudotuberculosis , Animais , Coelhos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligantes , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Yersinia pseudotuberculosis/metabolismoRESUMO
Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.
Assuntos
Proteínas de Saccharomyces cerevisiae , Sorghum , Animais , Humanos , Sorghum/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Saccharomyces cerevisiae , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismoRESUMO
Objetivo: identificar os desafios e as possibilidades de coparticipação das puérperas e dos acompanhantes no cuidado seguro na maternidade. Materiais e Métodos: estudo qualitativo realizado com 23 puérperas e 11 acompanhantes em uma maternidade de Belo Horizonte, entre março e julho de 2019. Os dados foram coletados por meio de entrevistas com roteiros semiestruturados e submetidos à análise de conteúdo temática, segundo o referencial teórico da segurança do paciente. Resultados: emergiram duas categorias: contribuição da puérpera e do acompanhante para o cuidado seguro; e desafios e contribuições para o alcance da coparticipação das puérperas e dos acompanhantes na segurança do paciente. Conclusões: acompanhantes e puérperas se reconhecem como coparticipantes na promoção da segurança do paciente, porém foi observada a falta de conhecimento e estímulo em relação à participação desses atores. Salienta-se a importância de utilizar tecnologias educativas para incluí-los como parceiros ativos na segurança do paciente.(AU)
Objective: to identify the challenges and possibilities of co-participation of puerperal women and companions in safe maternity care. Materials and Methods: qualitative study carried out with 23 mothers and 11 companions in a maternity hospital in Belo Horizonte, between March and July 2019. Data were collected through interviews with semi-structured scripts and submitted to thematic content analysis, according to the theoretical framework of patient safety. Results: two categories emerged: contribution of the puerperal woman and the companion for safe care; and challenges and contributions to achieving co-participation of puerperal women and companions in patient safety. Conclusions: companions and puerperal women recognize themselves as co-participants in promoting patient safety, however, a lack of knowledge and encouragement regarding the participation of these actors was observed. The importance of using educational technologies to include them as active partners in patient safety is highlighted.(AU)
Objetivo: identificar los desafíos y posibilidades de la coparticipación de puérperas y acompañantes en la atención a la maternidad segura. Materiales y Métodos: estudio cualitativo realizado con 23 puérperas y 11 acompañantes en una maternidad de Belo Horizonte, entre marzo y julio de 2019. Los datos fueron recogidos a través de entrevistas con guiones semiestructurados y sometidos a análisis de contenido temático de acuerdo con el marco teórico de la seguridad del paciente. Resultados: surgieron dos categorías: Contribución de la puérpera y del acompañante para el cuidado seguro; Desafíos y aportes para lograr la coparticipación de las puérperas y acompañantes en la seguridad del paciente. Conclusiones: los acompañantes y puérperas se reconocen como copartícipes en la promoción de la seguridad del paciente, pero falta conocimiento y estímulo en cuanto a la participación de estos actores. Se destaca la importancia de utilizar tecnologías educativas para incluirlos como socios activos en la seguridad del paciente.(AU)
Assuntos
Feminino , Gravidez , Adulto , Participação do Paciente , Acompanhantes Formais em Exames Físicos , Segurança do Paciente , Fatores Socioeconômicos , Família , Saúde Materno-Infantil , Pesquisa QualitativaRESUMO
Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.
Assuntos
Acrossomo , Proteínas SNARE , Humanos , Masculino , Acrossomo/metabolismo , Exocitose/fisiologia , Sêmen/metabolismo , Proteínas SNARE/metabolismo , Espermatozoides/metabolismoRESUMO
RESUMO Objetivo: Verificar a prevalência e identificar os fatores associados à ausência do acompanhante de parto em mulheres no sul do Brasil. Métodos: Trata-se de um estudo transversal, realizado com 466 parturientes, pertencentes a uma coorte de mulheres da zona urbana da cidade de Pelotas, RS. Aos 18 meses pós-parto, foi aplicado um questionário estruturado com dados sociodemográficos, gestacionais e questões relacionadas ao parto. Foi realizada regressão logística para ajustes de possíveis fatores de confusão. Resultados: A prevalência da ausência de acompanhante de parto entre as mulheres foi de 22,3%. As parturientes com até 8 anos de estudo (RP=2,0 [IC95% 1,1-3,8]), que não viviam com um companheiro (RP=2,3 [IC95% 1,2-4,3]), que realizaram o pré-natal no setor público (RP=1,9 [IC95% 1,0-3,7]) e que tiveram um parto via cesárea (RP=6,0 [IC95% 2,9-12,4]) apresentaram maior probabilidade de ausência de acompanhante de parto. Conclusão: Os resultados apontam evidências relevantes para o seguimento da verificação da presença do acompanhante de parto no sul do Brasil, indicando a necessidade de melhor aproveitamento e adesão desta prática. Além disso, a lei que aprova a presença do acompanhante de parto no Brasil parece não estar sendo colocada em prática de modo integral, desrespeitando um direito das parturientes e impactando nos benefícios para a saúde materno-infantil.
ABSTRACT Objective: To verify the prevalence and identify the factors associated with the absence of birth companions among women in Southern Brazil. Methods: This is a cross-sectional study carried out with 466 parturient women in a cohort of women from the urban area of the city of Pelotas, RS. At 18 months postpartum, a structured questionnaire was applied with sociodemographic, gestational data and questions related to childbirth. Logistic regression was performed to adjust for possible confounding factors. Results: The prevalence of the absence of a birth companion among women was 22.3%. Parturient women with up to 8 schooling years (PR=2.0 [95%CI 1.1-3.8]), who did not live with a partner (PR=2.3 [95%CI 1.2-4.3]), who performed their prenatal care in the public sector (PR=1.9 [95%CI 1.0-3.7]) and who had a cesarean delivery (PR=6.0 [95%CI 2.9-12.4]) were more likely to not have had a birth companion. Conclusion: The results shows relevant evidence for the verification of the presence of a companion in Southern Brazil, indicating the need for better use and adherence to this practice. In addition, the law that approves the presence of the birth companion in Brazil does not seem to be being fully implemented, disrespecting a right of parturient women and impacting the benefits for for maternal and child health.
RESUMO
Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature.
Assuntos
Proteínas de Choque Térmico Pequenas , Phaseolus , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Chaperonas MolecularesRESUMO
G protein-coupled receptors (GPCRs) are plasma membrane proteins associated with an array of functions. Mutations in these receptors lead to a number of genetic diseases, including diseases involving the endocrine system. A particular subset of loss-of-function mutant GPCRs are misfolded receptors unable to traffic to their site of function (i.e. the cell surface plasma membrane). Endocrine disorders in humans caused by GPCR misfolding include, among others, hypo- and hyper-gonadotropic hypogonadism, morbid obesity, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, X-linked nephrogenic diabetes insipidus, congenital hypothyroidism, and familial glucocorticoid resistance. Several in vitro and in vivo experimental approaches have been employed to restore function of some misfolded GPCRs linked to endocrine disfunction. The most promising approach is by employing pharmacological chaperones or pharmacoperones, which assist abnormally and incompletely folded proteins to refold correctly and adopt a more stable configuration to pass the scrutiny of the cell's quality control system, thereby correcting misrouting. This review covers the most important aspects that regulate folding and traffic of newly synthesized proteins, as well as the experimental approaches targeted to overcome protein misfolding, with special focus on GPCRs involved in endocrine diseases.
Assuntos
Doenças do Sistema Endócrino , Dobramento de Proteína , Membrana Celular/metabolismo , Doenças do Sistema Endócrino/metabolismo , Doenças do Sistema Endócrino/terapia , Humanos , Recém-Nascido , Mutação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
BACKGROUND: Endolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, including improvement of solubility and recovery from cell extracts. RESULTS: We report the expression of two endolysins encoded in the genome of phages infecting Staphylococcus aureus. Expression was optimized through changes in the concentration of the inducer and growth temperature during the expression. Usually, only 30-40% of the total endolysin was recovered in the soluble fraction. Co-expression of molecular chaperones (DnaK, GroEL) or N-term fusion tags endowed with increased solubility (DsbC, Trx, Sumo) failed to improve that yield substantially. Inclusion of osmolytes (NaCl, CaCl2, mannitol, glycine betaine, glycerol and trehalose) or tensioactives (Triton X-100, Tween 20, Nonidet P-40, CHAPS, N-lauroylsarcosine) in the cell disruption system (in the absence of any molecular chaperone) gave meager improvements excepted by N-lauroylsarcosine which increased recovery to 54% of the total endolysin content. CONCLUSION: This is the first attempt to systematically analyze methods for increasing yields of recombinant endolysins. We herein show that neither solubility tags nor molecular chaperones co-expression are effective to that end, while induction temperature, (His)6-tag location and lysis buffer additives (e.g. N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins.
Assuntos
Bacteriófagos , Escherichia coli , Bacteriófagos/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Staphylococcus aureus/metabolismoRESUMO
In a previous in vitro study, dihydropyrimidinone-derived selenoesteres demonstrated antioxidant properties, metal chelators and inhibitory acetylcholinesterase (AChE) activity, making these compounds promising candidates for Alzheimer's Disease (AD) treatment. However, these effects have yet to be demonstrated in an in vivo animal model; therefore, this study aimed to evaluate the safety and efficacy of eight selenoester compounds in a Caenorhabditis elegans model using transgenic strains for amyloid-beta peptide (Aß) aggregation. The L1 stage worms were acutely exposed (30 min) to the compounds at concentrations ranging from 5 to 200 µM and after 48 h the maintenance temperature was increased to 25 ° C for Aß expression and aggregation. After 48 h, several parameters related to phenotypic manifestations of Aß toxicity and mechanistic elucidation were analyzed. At the concentrations tested no significant toxicity of the compounds was found. The selenoester compound FA90 significantly reduced the rate of paralyzed worms and increased the number of swimming movements compared to the untreated worms. In addition, FA90 and FA130 improved egg-laying induced by levamisole and positively modulated HSP-6 and HSP-4 expression, thereby increasing reticular and mitochondrial protein folding response in C. elegans, which could attenuate Aß aggregation in early exposure. Therefore, our initial screening using an alternative model demonstrated that FA90, among the eight selenoesters evaluated, was the most promising compound for AD evaluation screening in more complex animals.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Pirimidinonas/farmacologia , Acetilcolinesterase/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Caenorhabditis elegans , Modelos Animais de Doenças , Levamisol/farmacologia , Fármacos Neuroprotetores/efeitos adversos , Organismos Geneticamente Modificados , Compostos Organosselênicos/efeitos adversos , Oviposição/efeitos dos fármacos , Pirimidinonas/efeitos adversosRESUMO
ABSTRACT Objective To estimate the prevalence of violation of the rights of the companion during the hospitalization of the woman for childbirth. Method Cross-sectional study conducted in public maternity hospitals in Florianopolis between 2015 and 2016, with data from individual interview with 1.145 companions. Prevalence ratio and Pearson's chi-square test were applied in the analysis. Results Women (92.8%), who received prenatal care (93.1%) and were unaware of the companions' law (92.7%) suffered more violation of rights. Not having received written guidance (93.6%), not identifying the health professional (65.0%) and not being encouraged to participate in care (55.9%) were violated rights. Welcoming and communicating with the team were the care aspects that most violated the rights of the companion. Conclusion The high prevalence of violation of rights demonstrates the disrespect and the need to value companions of choice.
RESUMEN Objetivo Estimar la prevalencia de violación de los derechos de lo acompañante durante la hospitalización de la mujer para el parto. Método Estudio transversal, realizado en maternidades públicas de Florianopolis, entre 2015 y 2016, con datos de entrevista individual con 1.145 acompañantes. La razón de prevalencia y la prueba de chi cuadrado de Pearson se aplicaron en el análisis. Resultados Las mujeres (92,8%), que siguieron la atención prenatal (93,1%) y desconocían la ley del acompañante (92,7%) sufrieron más violaciones de derechos. No haber recibido orientación escrita (93,6%), no identificar al profesional asistente (65,0%) y no ser alentado a participar en la atención (55,9%) fueron derechos violados. Acoger y comunicarse con el equipo fueron los aspectos de cuidado que más vulneraron los derechos del acompañante. Conclusión La alta prevalencia de violación de derechos demuestra la falta de respeto y la necesidad de valorar al acompañante.
RESUMO Objetivo Estimar a prevalência de violação de direitos do acompanhante durante a internação da mulher para o parto. Método Estudo transversal, conduzido em maternidades públicas de Florianópolis, entre 2015 e 2016, com dados de entrevista individual com 1.145 acompanhantes. Na análise, aplicou-se cálculo de razão de prevalência e teste qui-quadrado de Pearson. Resultados Mulheres (92,8%), que acompanharam o pré-natal (93,1%) e desconheciam a lei do acompanhante (92,7%) sofreram mais violação de direitos. Não ter recebido orientação escrita (93,6%), não ter identificado o profissional assistente (65,0%) e não ter sido estimulado a participar do cuidado (55,9%) foram direitos violados. O acolhimento e a comunicação com a equipe foram os aspectos assistenciais que mais infringiram direitos do acompanhante. Conclusão A elevada prevalência de violação de direitos demonstra o desrespeito e a necessidade de valorização do acompanhante de parto.